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Title:
MIR-17~92 AS THERAPEUTIC OR DIAGNOSTIC TARGET OF MOTOR NEURON (MN) DEGENERATION DISEASES
Document Type and Number:
WIPO Patent Application WO/2019/195304
Kind Code:
A1
Abstract:
The present disclosure relates to mir-17~92 as a candidate therapeutic or diagnostic target of motor neuron (MN) degeneration diseases. Expression of mir-17~92 is sustained throughout adulthood in spinal MNs and specifically decreases before the onset of MN loss in SOD1 G93A mice. Accordingly, mir-17~92 can be used as a candidate therapeutic target for ALS.

Inventors:
CHEN, Jun-An (128 Academia Road, Section 2 Nankan, Taipei Taiwan 115, 115, TW)
PENG, Kuan-Chih (128 Academia Road, Section 2 Nankan, Taipei Taiwan 115, 115, TW)
TUNG, Ying-Tsen (128 Academia Road, Section 2 Nankan, Taipei Taiwan 115, 115, TW)
Application Number:
US2019/025406
Publication Date:
October 10, 2019
Filing Date:
April 02, 2019
Export Citation:
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Assignee:
ACADEMIA SINICA (128 Academia Road, Section 2 Nankan, Taipei Taiwan 115, 115, TW)
SHIH, Ming-Che (4905 Tara Ter, Culver City, CA, 90230-4331, US)
International Classes:
A61K31/7105; A61K9/00; A61K9/51; A61K47/46; A61P21/00; A61P25/00; C12Q1/6883
Other References:
TUNG, Y.-T. ET AL.: "Mir-17-92 governs motor neuron subtype survival by mediating nuclear PTEN", CELL REPORTS, vol. 11, no. 8, 26 May 2015 (2015-05-26), pages 1305 - 1318, XP055640958
ZOU, Z. Y. ET AL.: "Screening for C9orf72 repeat expansions in Chinese amyotrophic lateral sclerosis patients", NEUROBIOLOGY OF AGING, vol. 34, no. 6, 20 December 2012 (2012-12-20) - June 2013 (2013-06-01), pages 1710.e5 - 1710.e6, XP055640964
TOLI, D. ET AL.: "Modeling amyotrophic lateral sclerosis in pure human iPSc -derived motor neurons isolated by a novel FACs double selection technique", NEUROBIOLOGY OF DISEASE, vol. 82, 21 June 2015 (2015-06-21), pages 269 - 280, XP055640968
HOU, L. ET AL.: "Screening of SOD1, FUS and TARDBP genes in patients with amyotrophic lateral sclerosis in central-southern china", SCIENTIFIC REPORTS, vol. 6, no. 1, 9 August 2016 (2016-08-09), pages 1 - 7, XP055640974
CUI, Q.-L. ET AL.: "Regulation of miRNA 219 and miRNA clusters 338 and 17-92 in oligodendrocytes", FRONTIERS IN GENETICS, vol. 3, no. 46, 28 March 2012 (2012-03-28), pages 1 - 13, XP055640977
Attorney, Agent or Firm:
NORTON, Vicki, G. (Duane Morris LLP, 750 B Street Suite 290, San Diego CA, 92101-4681, US)
Download PDF:
Claims:
Claims

What is claimed is:

1. A method of treating or preventing a motor neuron degeneration disease in a subject or ameliorating motor deficits and prolonging lifespan of a subject with motor neuron degeneration disease, comprising administering to the subject a therapeutically or prophylactically effective amount of one or more genes or transgenes encoding members of mi croRNA- 17-92 cluster or one or more members of mi croRNA- 17-92 cluster.

2. The method of Claim 1, wherein the administration comprises delivering one or more members of microRNA- 17-92 cluster into central nervous system of the subject.

3. The method of Claim 1, wherein the administration comprises delivering one or more members of microRNA- 17-92 cluster into spinal cord of the subject.

4. The method of Claim 1, wherein the treatment or prevention of the motor neuron degeneration disease is through overexpression of the one or more members of microRNA- 17-92 cluster in SOD1G93A MNS.

5. The method of Claim 4, wherein the overexpression of the one or more members of microRNA- 17-92 cluster in SOD1G93A MNS causes a concomitant reduced level of nuclear

PTEN.

6. The method of Claim 1, wherein an early and selective down-regulation of mir-17~92 in SOD1G93A MNS precedes the onset of MN degeneration.

7. The method of Claim 6, wherein selective mir-17~92/nPTEN dysregulation occurs early in SOD1G93A LMC MNS and may confer the MN subtype vulnerability to ALS disease.

8. The method of Claim 1, which delays or prevents the onset of MN degeneration or reduces a risk for developing MN degeneration.

9. The method of Claim 1, wherein the MN degeneration is amyotrophic lateral sclerosis (ALS) or spinal muscular atrophies (SMA).

10. The method of Claim 9, wherein the ALS is familial ALS or sporadic ALS.

11. The method of Claim 1, wherein the member of microRNA- 17-92 cluster is mir-17, mir-17*, mir-18a, mir-19a, mir-19b, mir-20a, or mir-92a.

12. The method of Claim 1, wherein the gene or transgene is incorporated into or encapsulated in a carrier for administration or delivery.

13. The method of Claim 12, wherein the carrier is a viral vector or nanoparticle.

14. The method of Claim 13, wherein the viral vector is scAAV9, any serotypes of recombinant AAV (such as AAV1, AAV2, AAV9, AAV2/1, AAV2/5, AAV2/8, AAV2/9), or ientivirus

15. The method of Claim 1, wherein the transgene comprises a HI promoter, CMV promoter, RSV promoter, SV40 promoter, human b-actin promoter, human elongation factor- lapromoter or cytomegalovirus early enhancer/chicken b-actin promoter and one or more gene encoding members of microRNA- 17-92 cluster.

16. The method of Claim 1, which further comprises administering one or more inhibitory nucleic acids targeting SOD1 or MMP9, a transgene encoding a nuclear PTEN interfering peptide, an ER stress inhibitor, a stress granule blocker or a miRNA biogenesis activator.

17. The method of Claim 16, wherein the inhibitory nucleic acid is antisense oligonucleotides, siRNA compounds, single- or double-stranded RNA interference (RNAi) compounds such as siRNA compounds, modified bases/locked nucleic acids (LNAs), peptide nucleic acids (PNAs), or other oligomeric compounds or oligonucleotide mimetics which hybridize to at least a portion of the target SOD1 or MMP9.

18. The method of Claim 1, wherein the administration is intrathecal injection.

19 A method of screening a drug protecting motor neuron subtypes in ALS in vitro , comprising (i) providing a stem cell line harboring a transgene comprising a fALS gene and a reporter gene, (ii) culturing a drug with the cell line; (iii) directing differentiation of the cell to the motor neuron cells and expression; and (iv) determining the said drug is effective to protect motor neuron subtypes in ALS if a motor neuron subtype is generated from the stem cell line.

20. The method of Claim 19, wherein the stem cell line is an embryonic stem cell line or iPSC cell line.

21. The method of Claim 19, wherein the stem cell line is human stem cell or mouse stem cell.

22. The method of Claim 19, wherein the fALS gene is C9orf72 mutation containing expanded GGGGCC repeats, FUS-R521C, FUS-P525R, FUS-R521H, FUS-Q210H, FUS- R514G, FUS-C1574G, SOD1-G93A, SOD1-A4V, SOD1-D90A, SOD1-D91A, SOD1-H46R,

SOD1-A89V, SOD1-I113T, TDP43-A315T, TDP43-Q343R, TDP43-M337V, TDP43-N345K, TDP43-I383V or TDP43-M337V.

23. The method of Claim 19, wherein the reporter is a double reporter.

24. The method of Claim 23, wherein the double reporter is Hb9::RFP and FoxPl::GFP. 25. A method for diagnosis of motor neuron subtypes in ALS in a subject, comprising providing a biological sample and detecting the presence of one or more members of microRNA- 17-92 cluster or PTEN, wherein the presence of dysregulated microRNA- 17-92 members or nuclear PTEN is indicative of a diagnosis of motor neuron subtypes in ALS or a susceptibility to motor neuron subtypes in ALS in the subject.

26. A kit for diagnosing motor neuron subtypes in ALS or susceptibility to motor neuron subtypes in ALS in a subject, comprising one or more reagents for detecting the presence of one or more members of microRNA- 17-92 cluster or nuclear PTEN in a biological sample.

AMENDED CLAIMS

received by the International Bureau on 18 Sep 2019 (18.09.2019).

1. Cancelled.

2. The method of any one of Claims 27-30, wherein the administration comprises delivering one or more members of microRNA- 17-92 cluster into central nervous system of the subject.

3. The method of any one of Claims 27-30, wherein the administration comprises delivering one or more members of microRNA- 17-92 cluster into spinal cord of the subject.

4. The method of any one of Claims 27-30, wherein the treatment or prevention of the motor neuron degeneration disease is through overexpression of the one or more members of microRNA- 17-92 cluster in SOD1G93A MNS.

5. The method of Claim 4, wherein the overexpression of the one or more members of microRNA- 17-92 cluster in SOD1G93A MNS causes a concomitant reduced level of nuclear PTEN.

6. The method of any one of Claims 27-30, wherein an early and selective down-regulation of mir-17~92 in SOD1G93A MNS precedes the onset of MN degeneration.

7. The method of Claim 6, wherein selective mir-17~92/ nPTEN dysregulation occurs early in SOD1G93A LMC MNS and may confer the MN subtype vulnerability to ALS disease.

8. The method of any one of Claims 27-30, which delays or prevents the onset of MN degeneration or reduces a risk for developing MN degeneration.

9. The method of any one of Claims 27-30, wherein the MN degeneration is amyotrophic lateral sclerosis (ALS) or spinal muscular atrophies (SMA).

10. The method of Claim 9, wherein the ALS is familial ALS or sporadic ALS.

11. The method of any one of Claims 27-30, wherein the member of microRNA- 17-92 cluster is mir-17, mir-17*, mir-18a, mir-19a, mir-19b, mir-20a, or mir-92a.

12. The method of any one of Claims 27-30, wherein the gene or transgene is incorporated into or encapsulated in a carrier for administration or delivery.

13. The method of Claim 12, wherein the carrier is a viral vector or nanoparticle.

14. The method of Claim 13, wherein the viral vector is scAAV9, any serotypes of recombinant AAV (such as AAV1, AAV2, AAV9, AAV2/1, AAV2/5, AAV2/8, AAV2/9), or lenti virus.

15. The method of any one of Claims 27-30, wherein the transgene comprises a HI promoter, CMV promoter, RSV promoter, SV40 promoter, human b-actin promoter, human elongation factor- 1 «promoter or cytomegalovirus early enhancer/chicken b-actin promoter and one or more gene encoding members of microRNA- 17-92 cluster.

16. The method of any one of Claims 27-30, which further comprises administering one or more inhibitory nucleic acids targeting SOD1 or MMP9, a transgene encoding a nuclear PTEN interfering peptide, an ER stress inhibitor, a stress granule blocker or a miRNA biogenesis activator.

17. The method of Claim 16, wherein the inhibitory nucleic acid is antisense oligonucleotides, siRNA compounds, single- or double-stranded RNA interference (RNAi) compounds such as siRNA compounds, modified bases/locked nucleic acids (LNAs), peptide nucleic acids (PNAs), or other oligomeric compounds or oligonucleotide mimetics which hybridize to at least a portion of the target SOD1 or MMP9.

18. The method of any one of Claims 27-30, wherein the administration is intrathecal injection.

19. A method of screening a drug protecting motor neuron subtypes in ALS in vitro, comprising (i) providing a stem cell line harboring a transgene comprising a fALS gene and a reporter gene,

(ii) culturing a drug with the cell line;

(iii) directing differentiation of the cell to the motor neuron cells and expression; and

(iv) determining the said drug is effective to protect motor neuron subtypes in ALS if a motor neuron subtype is generated from the stem cell line.

20. The method of Claim 19, wherein the stem cell line is an embryonic stem cell line or iPSC cell line.

21. The method of Claim 19, wherein the stem cell line is human stem cell or mouse stem cell.

22. The method of Claim 19, wherein the fALS gene is C9orf72 mutation containing expanded GGGGCC repeats, FUS-R521C, FUS-P525R, FUS-R521H, FUS-Q210H, FUS-R514G, FUS-C1574G, SOD1-G93A, SOD1-A4V, SOD1-D90A, SOD1-D91A, SOD1-H46R, SOD1-A89V, SOD1-I113T, TDP43-A315T, TDP43-Q343R, TDP43-M337V, TDP43-N345K, TDP43-I383V or TDP43-M337V.

23. The method of Claim 19, wherein the reporter is a double reporter.

24. The method of Claim 23, wherein the double reporter is Hb9::RFP and FoxPl : :GFP.

25. A method for diagnosis of motor neuron subtypes in ALS in a subject, comprising providing a biological sample and detecting the presence of one or more members of micro RNA- 17-92 cluster or PTEN, wherein the presence of dysregulated microRNA- 17-92 members or nuclear PTEN is indicative of a diagnosis of motor neuron subtypes in ALS or a susceptibility to motor neuron subtypes in ALS in the subject.

26. A kit for diagnosing motor neuron subtypes in ALS or susceptibility to motor neuron subtypes in ALS in a subject, comprising one or more reagents for detecting the presence of one or more members of microRNA- 17-92 cluster or nuclear PTEN in a biological sample.

27. Use of a therapeutically or prophylactically effective amount of one or more genes or transgenes encoding members of microRNA- 17-92 cluster or one or more members of microRNA- 17-92 cluster for treatment or prevention of a motor neuron degeneration disease in a subject.

28. Use of a therapeutically or prophylactically effective amount of one or more genes or transgenes encoding members of microRNA- 17-92 cluster or one or more members of microRNA- 17-92 cluster for ameliorating motor deficits and prolonging lifespan of a subject with motor neuron degeneration disease.

29. Use of a therapeutically or prophylactically effective amount of one or more genes or transgenes encoding members of microRNA- 17-92 cluster or one or more members of microRNA- 17-92 cluster in the manufacture of a medicament for the treatment or the prevention of a motor neuron degeneration disease in a subject.

30. Use of a therapeutically or prophylactically effective amount of one or more genes or transgenes encoding members of microRNA- 17-92 cluster or one or more members of microRNA- 17-92 cluster in the manufacture of a medicament for ameliorating motor deficits and prolonging lifespan of a subject with motor neuron degeneration disease.

Description:
MIR-17-92 AS THERAPEUTIC OR DIAGNOSTIC TARGET OF MOTOR NEURON

(MN) DEGENERATION DISEASES

Field of the Invention

[ 0001 ] The present disclosure relates to the field of microRNA (miRNA), in particular mir- 17-92 as a candidate therapeutic or diagnostic target of motor neuron (MN) degeneration diseases.

Background of the Invention

[ 0002 ] ALS, also known as Lou Gehrig's dis ease, is a devastating adult-onset neurodegenerative disease that attacks upper and lower motor neurons. A progressive and ultimately fatal muscle paralysis ensues, usually causing death within 2 to 5 years of disease onset. ALS is mostly sporadic, but approximately 10% of cases are familial. The incidence rate is approximately 2-3 cases per 100,000 people per year worldwide. In about 75% of ALS patients, the distal limb-innervating MNs, especially the fast-twitch fatigable (FF) MNs that control rapid limb movements, are usually the first subtype of MNs to undergo degeneration, so patients initially experience weakness or atrophy in the arms or legs. Other MN subtypes are subsequently affected and, within 3 to 5 years, patients suffer complete paralysis and ultimately die due to respiratory failure. The only marketed drug (Riluzole) for treating ALS has modest effects, improving muscle function but having strong side-effects and no significant impact on patient survival. Recently, a new drug (Edaravone) was approved by the US Food & Drug Administration (FDA), but its efficacy remains to be evaluated. Thus far, there is no effective cure for this fatal disease.

[ 0003 ] Pathogenic mutations in several genes have been linked to familial and sporadic ALS, including SOD1, TARDBP, FUS/TLS, VAPB, OPTN and others. Dominant mutations in the superoxide dismutase 1 ( SOD1 ) gene account for 15-20% of familial ALS (fALS). Although the exact pathological role of SOD1 in ALS remains unclear, a series of transgenic mice carrying fALS SOD1 mutants have been established, which recapitulate the pathological phenotype of ALS progression, including limb weakness as the first sign of disease onset, and provide a valuable model for studying ALS mechanisms. Cumulative studies in these fALS-SODl transgenic models suggest that dysregulation of multiple cellular mechanisms cooperatively contribute to ALS progression. One of the most intensively studied pathways is endoplasmic reticulum (ER) stress (Kikuchi H, et al. (2006) Spinal cord endoplasmic reticulum stress associated with a microsomal accumulation of mutant superoxide dismutase-1 in an ALS model. Proceedings of the National Academy of Sciences of the United States of America 103(15):6025-

6030), which is more prominently observed in vulnerable FF-MNs. RNA metabolism has also been linked to ALS pathology, as many of the identified disease-associated genes encode for RNA-binding proteins, such as TARDBP, FUS, and HNRNPA1. Interestingly, these disease- causing genes also participate in miRNA biogenesis and metabolism. Recent studies have further revealed that dysregulation of miRNA expression is manifested in ALS mouse models and patients ( Eitan C & Hornstein E (2016) Vulnerability of microRNA biogenesis in FTD-ALS. Brain Res 1647:105-111 ).

[ 0004 ] MicroRNAs (miRNAs) are endogenous noncoding small RNAs that play critical roles in cell fate specification and neuronal survival in the spinal cord. A recent study revealed that pathogenic ALS-causing mutations are sufficient to inhibit Dicer complex activation, which is mediated by cytosolic macromolecular assembly of stress granules (SGs), leading to global down-regulation of miRNA biogenesis. Nevertheless, the precise functions and sets of miRNAs involved in ALS have not been fully established. Although loss of the muscle-specific miRNA, mir-206 , can accelerate the onset of MN loss in a fALS -SOD1G93A mouse model (Williams AH et al. (2009) MicroRNA-206 delays ALS progression and promotes regeneration of neuromuscular synapses in mice. Science 326(5959): 1549-1554), it is still unclear if MN-specific miRNAs also participate in cell-autonomously regulating MN survival. Conditional loss of Dicer activity to block miRNA biogenesis in MN lineages of mouse embryos leads to selective degeneration of a subset of MNs, including limb-innervating MNs ( Chen JA & Wichterle H (2012) Apoptosis of limb innervating motor neurons and erosion of motor pool identity upon lineage specific dicer inactivation. Frontiers in neuroscience 6:69). mir-17~92 is an MN-enriched miRNA cluster that is essential for controlling embryonic limb-innervating MN (LMC-MN) survival ( Tung YT, et al. (2015) Mir-17~92 Governs Motor Neuron Subtype Survival by Mediating Nuclear PTEN. Cell reports 11(8):1305-1318). This protective effect is mediated by inhibition of PTEN monoubiquitination by Nedd4-2 and Ndfipl, subsequent nuclear PTEN monoubiquitination by Nedd4-2 and Ndfipl, and subsequent nuclear PTEN translocalization.

Summary of the Invention

[ 0005 ] The present disclosure surprisingly found that expression of mir-17~92 is sustained throughout adulthood in spinal MNs and that it specifically decreases before the onset of MN loss in S0D1 G93A mice. This timing is concomitant with accumulation of nuclear PTEN in the spinal MNs of S0D1 G93A mice. Importantly, overexpression of mir-17~92 in SOI) l' ' MNs by either genetic approaches or adult scAAV9 delivery can significantly delay motor deficits and prolong lifespan. These findings define mir-17~92 as a candidate therapeutic target for ALS.

[ 0006 ] The present disclosure provides a method of treating or preventing a motor neuron degeneration disease in a subject, comprising administering to the subject a therapeutically or prophylactically effective amount of one or more genes or transgenes encoding members of microRNA- 17-92 cluster.

[ 0007 ] The present disclosure also provides a method of ameliorating motor deficits and prolonging lifespan of a subject with motor neuron degeneration disease, comprising administering to the subject an effective amount of one or more genes or transgenes encoding members of microRNA- 17-92 cluster. In one embodiment, the method can delay or prevent the onset of MN degeneration or reduce the risk of developing MN degeneration.

[ 0008 ] Certain embodiments include delivering one or more members of microRNA- 17-92 cluster into central nervous system of the subject. Particularly, the one or more members of microRNA- 17-92 cluster are delivered into spinal cord of the subject.

[ 0009 ] The present disclosure also provides a method of screening a drug protecting motor neuron subtpyes in ALS in vitro , comprising (i) providing a stem cell line harboring a transgene comprising a fALS gene and a reporter gene, (ii) culturing a drug with the cell line; (iii) directing differentiation of the cell to the motor neuron cells and expression; and (iv) determining the said drug is effective to protect motor neuron subtypes in ALS if a motor neuron subtype is generated from the stem cell line.

[ 0010 ] The present disclosure further provides a method for diagnosis of motor neuron subtypes in ALS in a subject, comprising providing a biological sample and detecting the presence of one or more members of microRNA- 17-92 cluster or nuclear PTEN, wherein the presence of dysregulated microRNA- 17-92 members or nuclear PTEN is indicative of a diagnosis of motor neuron subtypes in ALS or a susceptibility to motor neuron subtypes in ALS in the subject. [ 0011 ] The present disclosure further provides a kit for diagnosing motor neuron subtypes in ALS or susceptibility to motor neuron subtypes in ALS in a subject, comprising one or more reagents for detecting the presence of one or more members of microRNA- 17-92 cluster or nuclear PTEN in a biological sample.

Brief Description of the Drawings

[ 0012 ] Figures 1 A to 1K show that mir-17~92 is required to maintain adult spinal MNs. (Fig. 1A) Illustration of how mir- 17-92 controls PTEN subcellular localization by directly targeting the Nedd4-2 E3 ubiquitin ligase and its adaptor, Ndfipl, thus inhibiting pro-apoptotic nuclear PTEN (nPTEN) accumulation in developing LMC-MNs (Tung et al., 2015). This study further aims to reveal the potential involvement of the wz>-77~92/nPTEN axis in MN degeneration in ALS. (Fig. 1B) Representative in situ hybridization of miR-17 expression in ChAT on MNs of lumbar spinal cords at P40. Dashed lines demarcate spinal cord margin and grey matter. Scale bar, 50 pm. (Fig. 1C) Schematic illustration of neuron distribution in adult spinal cord. Individual neuron cell bodies in the dorsal non-MN region and ventral -lateral MN region were laser capture microdissected and subjected to RNA extraction. (Fig. 1D) Representative frozen sections of the hemi-ventral horn region, stained with Azure B dye (see STAR METHODS). Numbers in the right panel are automatically assigned by the microdissection software during microdissection. Scale bar, 50 pm. LCM, laser capture microdissection. (Fig. 1E and Fig. 1F) qPCR analysis of ChAT and individual members of the mir-17~92 cluster in laser-microdissected cells. Dorsal non-MNs were captured as a control (mean ± s.d., n = 4 mice. Two-tailed t-tests were performed between groups of interest. *P<0.05). FC: fold change. (Fig. 1G and Fig. II) Immunostaining and quantification of ChAT on MN numbers in lumbar hemi-ventral horn sections of mir-17~92 MNb mice at P30 reveal a significant loss of MNs. (mean ± s.d., n ³ 3 mice, *P<0.05, two-tailed t-tests). (Fig. 1H and Fig. 1J) NMJs of gastrocnemius (GA) muscle at P30. mir-17~92 MNL mice manifest a significant reduction of NMJ innervations. a-BTC: a- bungarotoxin to detect nicotinic acetylcholine receptors (AChR). (mean ± s.d., n = 3 mice, *P<0.05, two-tailed t-tests). (Fig. 1K) Reduced neuromuscular function in mir- 17~92 ' 1 nD mice, as revealed by evoked CMAPs in the GA muscle after stimulation of the sciatic nerve. Quantification of CMAPs was determined by calculating the maximum peak-to-peak values (mean ± s.d, n = 6 mice, *P<0.05, two-tailed t-tests).

[ 0013 ] Figures 2A to 2J show dysregulation of mir-17~92/nPTEN in presymptomatic S0D1 G93A MNs. (Fig. 2A and 2B) Immunostaining and quantification of ChAT on MN numbers in sections of the lumbar hemi -ventral horn region reveal a significant loss of S0D1 G93A MNs starting from P100. White dashed lines demarcate the spinal cord margin. Scale bar, 50 pm. (n > 4 mice, mean ± s.d., *P<0.05, two-way ANOVA). (Fig. 2C and 2D) Representative in situ hybridization of miR-17 expression in hemi -ventral lateral ChAT on MNs of lumbar spinal cords at different stages in (Fig. 2C) and quantification of miR-17 in (Fig. 2D). Note that miR-17 expression in S0D1 G93A MNs started to decline at P80, before an obvious decrease in MNs in S0D1 G93A . Scale bar, 50 pm. (n ³ 4 mice, mean ± s.d., *P<0.05, two-way ANOVA). (Fig. 2E) Schematic illustration of laser captures of frozen sections of ventral-lateral horn regions from control and S0D1 G93A mice (see STAR METHODS). Only large MNs with a regular shape (intact MNs) were microdissected. (Fig. 2F to 2H) qPCR analyses of laser-captured MNs from S0D1 G93A mice at P100. (Fig. 2F) The comparable expression of ChAT confirms that only intact MNs were collected. (Fig. 2G) S0D1 G93A MNs exhibit down-regulation of mir-17~92 cluster members before MN loss. (Fig. 2H) Targets of mir-17~92 , including PTEN , Ndfipl and Nedd4- 2, are up-regulated in S0D1 G93A MNs. Data is shown as fold change (FC) of S0D1 G93A MNs relative to Ctrl MNs. (mean ± s.d., n = 4 mice, *P<0.05, two-tailed t-tests). (Fig. 21 and 2J) Immunostaining of PTEN in ChAT on MNs of the lumbar spinal cord at different stages in (Fig. 21). High magnifications of the highlighted PTEN cells in the yellow square are shown in the right-most panels. White dashed lines depict MN nuclei. Scale bar is 10 pm. (Fig. 2J) Quantification of nucleus-to-cytosol ratios of PTEN intensities in ChAT on MNs indicates an accumulation of nuclear PTEN in S0D1 G93A MNs at P100. (mean ± s.d., n ³ 4 mice, *P<0.05, two-way ANOVA).

[ 0014 ] Figures 3A to 3G show that SOD1G93A LMC-MNS are more susceptible to degeneration and exhibit early reduction of mir-17~92. (Fig. 3 A) Schematic illustration of Ctrl and S0D1 G93A ESC differentiation into postmitotic MNs with Hb9::RFP; Foxpl ::GFP double reporters (see STAR METHODS for details). To accelerate MN degeneration, cyclopiazonic acid (CPA) was introduced into MNs and the survival rate of MN subtypes was determined by flow cytometry. MNs treated with the vehicle (DMSO) were assessed as non-stressed controls. (Fig. 3B and 3C) Survival rates of each MN subtype from Ctrl (blue) and S0D1 G93A (red) Hb9::RFP; Foxpl : :GFP ESCs were quantified by flow cytometry every 6 hrs after CPA or vehicle treatment. Percentages of each MN subpopulation at indicated time-points are normalized to that at 0 hr (i.e. right before treatment). S0D1 G93A LMC-MNs undergo dramatic cell loss from 30 hr to 48 hr post-CPA treatment compared to treated Ctrl LMC-MNs. In contrast, both Ctrl and S0D1 G93A non-LMC-MNs are more resistant to CPA treatment (mean ± s.d. from n = 3 independent experiments. Detailed statistical results are shown in Tables S2 and S3). (Fig. 3D to 3G). FACS-purified MN subtypes at 24 hr post-treatment. Expression of mir-17~92 members (Fig. 3D and 3E) and their targets PTEN, Nedd4-2, Ndfipl) (Fig. 3F and 3G) in each population was determined by qPCR. miRNA/mRNA levels were normalized to the corresponding Ctrl subtype. Dark purple highlights CPA-treated groups, whereas light purple denotes groups treated with vehicle alone (mean ± s.d. of at least 3 independent experiments, *P<0.05, two tailed t-tests).

[ 0015 ] Figures 4A to 4E show that S0D1 G93A LMC-MNs manifest early nuclear translocation of PTEN, resulting in their vulnerability to ALS. (Fig. 4A) Immunostaining of PTEN subcellular localization in dissociated MNs after 24 hrs of CPA treatment. Red dashed lines depict the borders of MN nuclei. Scale bar, 10 pm. (Fig. 4B) Quantification of nucleus-to- cytosol ratios of PTEN average intensities in GFP off RFP on non-LMC-MNs and GFP on RFP on LMC-MNs. (mean ± s.d., n = 3 independent experiments, *P<0.05, two tailed t-tests). (Fig. 4C) Schematic illustration of the stages performed during in ovo electroporation in the embryonic spinal cord. (Fig. 4D) Immunostaining of lumbar spinal cord sections demonstrates a reduction in Foxpl 0n LMC MNs at the electroporated side of GFP-PTEN-NLS elec spinal cords, whereas Lhx3 on non-LMC neurons are more resistant to ectopic GFP-PTEN expression. (Fig. 4E) Quantification of Foxpl 0n LMC MNs and Lhx3 on non-LMC neurons. Data is shown as fold change of cell numbers in the electroporated side relative to the contralateral side (mean ± s.d., n = 5/6 embryos, *P<0.05, one-way ANOVA).

[ 0016 ] Figures 5 A to 5H show conserved dysregulation of the hsa-mir-17~92/nPTEN pathway in S0D1 +/L144F human iPSC-derived MNs. (Fig. 5A) Schematic depiction of CRISPR- Cas9-mediated gene correction. The genomic sequence surrounding the S0D1 +/L144F mutation (G-to-C mutation) is highlighted in red. The guide RNA recognition sequence is underlined. A 99 nucleotide single-stranded oligonucleotide containing the corrected G (highlighted in blue) was introduced as a template for homology-directed repair (HDR). (Fig. 5B) The corrected base (G/C - G/G) in the Ctrl- SODl +/+ line was confirmed by DNA sequencing. (Fig. 5C) Timeline reflecting the schedule for neurotrophic factor (NF) withdrawal. (Fig. 5D and 5E) ALS- S0D1 +/L144F LMC-MNs are more vulnerable to NF withdrawal compared to Ctrl -SOI) I LMC- MNs during long-term culture. (Fig. 5D) Immunostaining to reveal LMC-MN (SMI32 on , ISL1 on and FOXP1 on ) at indicated time points during NF withdrawal. (Fig. 5E) The survival rates of LMC-MNs were determined by quantification of FOXPl on ISLl on numbers at indicated time- points after NF withdrawal and were normalized to that before NF withdrawal (-NF day 0). (mean ± s.d., n=4 independent experiments, *P<0.05, two-way ANOVA). Scale bar, 100 pm. (Fig. 5F) Expression of hsa-mir-17~92 in long-term MN cultures at day 14 after NF withdrawal, as determined by qPCR. Data is shown as fold change (FC) of ALS-S0D1 +/L144F relative to Ctrl- SODl +/+ . (mean ± s.d. of at least four independent experiments, *P<0.05, two tailed t-tests). (Fig. 5G) Western blot indicating that protein levels of PTEN, NDFIP1 and NEDD4-2 are increased in S0D1 +/L144F MN culture at day 14 after NF withdrawal. a-Tubulin was included as a loading control. Quantitative data is shown as fold change (FC) of ALS-S0D1 +/L144F relative to Ctrl- SODl +/+ . (mean ± s.d., n=4 independent experiments, *P<0.05, two tailed t-tests). (Fig. 5H) Immunostaining to show subcellular localization of PTEN in FOXPl on ISLl on LMC-MNs at day 14 after NF withdrawal. Red dashed lines depict the borders of MN nuclei. Scale bar, 10 pm. Quantification of nucleus-to-cytosol ratios of PTEN average intensities (right panel) reveals elevated nuclear PTEN translocation in S0D1 +/L144F LMC-MNs. (mean ± s.d., n = 4 independent experiments, *P<0.05, two tailed t-tests).

[ 0017 ] Figures 6A to 6E show that survival of S0D1 +/L144F LMC-MNs can be extended by overexpression of hsa-mir- 17-92. (Fig. 6A) Schematic illustration of experimental timeline. iPSC~MN cultures at differentiation day 25 were subjected to overnight lentivirus (LV) infection to overexpress hsa-mir-17~92 or YFP as a control, followed by survival assay upon NF withdrawal from differentiation day 32. (Fig. 6B and 6C) qPCR analysis of infected MN cultures at the survival assay end-point (-NF day 28). (Fig. 6B) Lentiviral transduction results in ectopic and sustained overexpression of hsa-mir- 17-92. (Fig. 6C) Up-regulated PTEN , NDFIP1 and NEDD4-2 mRNA levels in stressed S0D1 +/L144F iPSC~MNs are suppressed by overexpression of hsa-mir- 17-92. Data is shown as fold change (FC) of each group relative to S0D1 +/L144F LV- YFP. (mean ± s.d., n=4 independent experiments, *P<0.05, one-way ANOVA). (Fig. 6D and 6E) Overexpression of hsa-mir-17~92 significantly increases S0D1 +/L144F LMC-MN survival upon NF withdrawal. (Fig. 6D) LV-infected LMC-MNs before and after NF withdrawal, as revealed by SMI32 and FOXP1 immunostaining. (Fig. 6E) LMC-MN survival rate was determined by quantification of LMC-MN (FOXPl on ISLl on ) numbers at day 28 of NF withdrawal and were normalized to LMC-MN numbers before NF withdrawal (mean ± s.d., n=4/5 independent experiments, *P<0.05, **P<0.0l, one-way ANOVA).

[ 0018 ] Figures 7A to 7H show that overexpression of mir-17~92 in MNs delays the onset of disease and extends lifespans of S0D1 G93A mice. (Fig. 7A) Genotypes of Ctrl and ALS mice, as well as the rescued ROSA26-loxp-STP-loxp (LSL)-mir- 17-92 in S0D1 G93A strain that overexpresses mir-17~92 in MNs by the Olig2 Cre/+ driver. (Fig. 7B and 7C) Immunostaining and quantification of ChAT on MN numbers in sections of the lumbar hemi-ventral horn region at P100 reveals a significant restoration of S0D1 G93A MN numbers. White dashed lines demarcate spinal cord margins. Scale bar, 50 pm. (n = 4 mice, mean ± s.d., *P<0.05, one-way ANOVA). (Fig. 7D and 7E) Immunostaining and quantification of PTEN subcellular localization in hemi- ventral lateral ChAT on MNs of the lumbar spinal cord at P100. Nuclear PTEN accumulation in S0D1 G93A MNs is reduced by mir-17~92 overexpression. High magnifications of the highlighted PTEN cells are shown in the right-most panels. White dashed lines depict MN nuclei. Scale bar is 10 mih. (mean ± s.d., n = 4 mice, *P<0.05, one-way ANOVA). (Fig. 7F) Roentgenograms of mice showing kyphosis phenotypes. Note that kyphosis is ameliorated in ALS; mir-17-92 MN~OE mice. Red dotted lines depict hind limb shape; black dotted lines depict mouse spine. (Fig. 7G) Rotarod test for motor performance. Latency to fall from an accelerating rotarod was determined and normalized to initial performance at P100 (as 100%). ALS; mir-17-92^-^ mice display improved performance from P120. (mean ± s.d., Ctrl (n = 5), ALS (n = 8), and ALS; mir- 17~92 MN E (n = 6) mice, *P<0.05 for comparing ALS and ALS; mir-17~92 MN OE mice by two- way ANOVA). (Fig. H) Kaplan-Meier survival curves reflect the survival of Ctrl (n = 32), ALS (n = 33), and ALS; mir-17-92 MN~OE (n = 22) mice. (****p < 0.0001 for comparing ALS and ALS; mir-17~92 MN E mice by log-rank Mantel-Cox test).

[ 0019 ] Figures 8 A to 8E show that gene therapy of S0D1 G93A mice by overexpressing mir- 17-92 at adulthood is sufficient to protect neuromuscular function and extend lifespan. (Fig. 8 A) Overview of the experimental strategy. Timeline shows the treatment schedule and stages for analyses. The schematic at left illustrates lumbar intrathecal injection (between L6 and Sl) of scAAV9 expressing the primary transcript of mir-17~92 or GFP as a control. (Fig. 8B) In situ hybridization of miR-17 in a P100 lumbar spinal cord in S0D1 G93A mice subjected to control scAAV9-GFP or scAW9-mir-17~92 injection. Scale bar, 50 pm. (Fig. 8C) qPCR analysis reveals a significant increase in mir-17-92 expression in the whole lumbar spinal cords of S0D1 G93A mice at P100 after scA A \9-mir- 17-92 injection compared to that of scAAV9-GFP injected SODl G93A mice (mean ± s.d., n=4, *P<0.05, two-tailed t-tests). (Fig. 8D) Neuromuscular function before and after scAAV9 injection, as assayed by evoked CMAPs in the GA muscle after stimulation of the sciatic nerve. The averages of maximum peak-to-peak CMAP values from each group of indicated age are plotted. The CMAP amplitude is already reduced in S0D1 G93A mice at the time of scAAV9 injection and gradually reduces further over time, whereas overexpression of mir- 17-92 significantly ameliorates neuromuscular function at P 120 and P140. (mean ± s.d., n=4~7 for each group, *P<0.05 for comparing scA A \9-mir- 17~92 injected and scAAV9-GFP injected mice by two-way ANOVA). (Fig. 8E) The lifespans of S0D1 G93A mice are prolonged by -14% following scAAV9 delivery of mir-17~92. Kaplan- Meier survival curves reflect the survival of Ctrl; AAV9-mir-17~92 (n = 13), ALS; AAV9 -GFP (n= 15), and ALS; ANV9-mir-l 7~92 (n =17) mice. (***P < 0.0005 for comparing ALS; AAV9- GFP and ALS; AAN9-mir-17~92 mice by log-rank Mantel-Cox test).

Detailed Description of the Invention

[ 0020 ] Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments described herein, some preferred methods, compositions, devices, and materials are described herein. However, before the present materials and methods are described, it is to be understood that this invention is not limited to the particular molecules, compositions, methodologies or protocols herein described, as these may vary in accordance with routine experimentation and optimization. It is also to be understood that the terminology used in the description is for the purpose of describing the particular versions or embodiments only, and is not intended to limit the scope of the embodiments described herein.

[ 0021 ] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[ 0022 ] As used herein and in the appended claims, the singular forms "a," "an" and "the" include plural references unless the context clearly dictates otherwise. [ 0023 ] As used herein interchangeably, the term a "miR gene product," "microRNA," "miRNA" or "miR" means a non-coding RNA between 18 and 25 nucleobases in length which hybridizes to and regulates the expression of a coding RNA.

[ 0024 ] As used herein, the terms "administration" and "administering" refer to the act of giving a drug, prodrug, or other agent, or therapeutic to a subject or in vivo, in vitro, or ex vivo cells, tissues, and organs.

[ 0025 ] As used herein, the term "at risk for developing" means a subject is predisposed to developing a condition or disease. In certain embodiments, a subject at risk for developing a condition or disease exhibits one or more symptoms of the condition or disease, but does not exhibit a sufficient number of symptoms to be diagnosed with the condition or disease. In certain embodiments, a subject at risk for developing a condition or disease exhibits one or more symptoms of the condition or disease, but to a lesser extent than required to be diagnosed with the condition or disease.

[ 0026 ] As used herein, the term "prevent the onset of means to prevent the development of a condition or disease in a subject who is at risk for developing the disease or condition. In certain embodiments, a subject at risk for developing the disease or condition receives treatment similar to the treatment received by a subject who already has the disease or condition.

[ 0027 ] As used herein, the term "delay the onset of means to delay the development of a condition or disease in a subject who is at risk for developing the disease or condition. In certain embodiments, a subject at risk for developing the disease or condition receives treatment similar to the treatment received by a subject who already has the disease or condition.

[ 0028 ] In one aspect, the present disclosure provides a method of treating or preventing a motor neuron degeneration disease in a subject, comprising administering to the subject a therapeutically or prophylactically effective amount of one or more genes or transgenes encoding members of microRNA- 17-92 cluster.

[ 0029 ] In another aspect, the present disclosure provides a method of ameliorating motor deficits and prolonging lifespan of a subject with motor neuron degeneration disease, comprising administering to the subject an effective amount of one or more genes or transgenes encoding members of microRNA- 17-92 cluster.

[ 0030 ] In some embodiments, the administration of the invention comprises delivering one or more members of microRNA- 17-92 cluster into central nervous system of the subject. In some embodiments, the one or more members of microRNA- 17-92 cluster are delivered into spinal cord of the subject.

[ 0031 ] In some embodiments, the treatment or prevention of the motor neuron degeneration disease is through overexpression of the one or more members of microRNA- 17-92 cluster in SOD1G93A MNs. In one embodiment, the overexpression of the one or more members of microRNA- 17-92 cluster in SOD1G93A MNS causes a concomitant reduced expression of PTEN and the E3 ligases, Nedd4-2 and Ndfipl. In one embodiment, an early and selective down- regulation of mir-17~92 in SODEGS I MNS precedes the onset of MN degeneration. Accordingly, mir-17~92/nPTEN manifests an MN-signature molecular change right before onset of MN degeneration in the SOD1G93A spinal cord. In one embodiment, selective mir-17~92/nPTEN dysregulation occurs early in SOD1G93A LMC MNS and may confer the MN subtype vulnerability to ALS disease.

[ 0032 ] In some embodiments, the method of the invention can delay or prevent the onset of MN degeneration or reduce the risk of developing MN degeneration. [ 0033 ] A motor neuron disease (MND) is any of several neurodegenerative disorders that selectively affect motor neurons, the cells that control voluntary muscles of the body. In some embodiments, the motor neuron degeneration disease described in the present disclosure includes amyotrophic lateral sclerosis (ALS) and spinal muscular atrophies (SMA). In some embodiments, the ALS is familial ALS or sporadic ALS.

[ 0034 ] In some embodiments, the members of microRNA- 17-92 cluster include, but are not limited to, mix- 17, mix-! 7*, mir~18a, mix- 19a, mir-!9b, mir-20a, and mir-92a. In one embodiment, the member of microRNA- 17-92 cluster is mir-l7.

[ 0035 ] In some embodiments, the gene or transgene of the present disclosure is incorporated into or encapsulated in a carrier for administration or delivery. In some embodiments, the carrier is a viral vector or nanoparticle. In some embodiments, the viral vector is scAAV9, any serotypes of recombinant AAV (such as AAV1, AAV2, AAV9, AAV2/1, AAV2/5, AAV2/8, AAV2/9), or lentivirus. Any promoter capable of driving microRNA- 17-92 cluster expression can be used in vector. In some embodiments, the promoter is HI, CMV, RSV, SV40, human b~ actin, human elongation factor-l a or cytomegalovirus early enhancer/chicken b-actin. In a certain embodiment, the transgene of the present disclosures comprises a CMV promoter and one or more gene encoding members of microRNA- 17-92 cluster.

[ 0036 ] In some embodiments, the method of the invention further comprises administering one or more inhibitory nucleic acids targeting SOD1 or MMP9, a transgene encoding a nuclear PTEN interfering peptide, an ER stress inhibitor, a stress granule blocker or a miRNA biogenesis activator.

[ 0037 ] Inhibitory nucleic acids useful in the present methods include antisense oligonucleotides, siRNA compounds, single- or double-stranded RNA interference (RNAi) compounds such as siRNA compounds, modified bases/locked nucleic acids (LNAs), peptide nucleic acids (PNAs), and other oligomeric compounds or oligonucleotide mimetics which hybridize to at least a portion of the target SOD1 or MMP9. In some embodiments, the inhibitory nucleic acids include antisense RNA, antisense DNA, chimeric antisense oligonucleotides, antisense oligonucleotides comprising modified linkages, interference RNA (RNAi), short interfering RNA (siRNA); a micro, interfering RNA (miRNA); a small, temporal RNA (stRNA); or a short, hairpin RNA (shRNA); small RNA-induced gene activation (RNAa); small activating RNAs (saRNAs), or combinations thereof.

[ 0038 ] For example, antisense oligonucleotides are typically designed to block expression of a DNA or RNA target by binding to the target and halting expression at the level of transcription, translation, or splicing. Antisense oligonucleotides of the present disclosure are complementary nucleic acid sequences designed to hybridize under stringent conditions to SOD1 or MMP9.

[ 0039 ] For example, the inhibitory nucleic acids used in the methods described herein comprise one or more modified bonds or bases. Modified bases include phosphorothioate, methylphosphonate, peptide nucleic acids, or locked nucleic acid (LNA) molecules. Preferably, the modified nucleotides are locked nucleic acid molecules, including alpha-L-LNAs. LNAs comprise ribonucleic acid analogues wherein the ribose ring is "locked" by a methylene bridge between the 2'-oxgygen and the 4'-carbon; i.e., oligonucleotides containing at least one LNA monomer.

[ 0040 ] For example, the nucleic acid sequence that is complementary to SOD1 or MMP9 can be an interfering RNA, including but not limited to a small interfering RNA ("siRNA") or a small hairpin RNA ("shRNA"). Methods for constructing interfering RNAs are well known in the art. [ 0041 ] The gene of the present disclosure or the agent described herein for in combination with the gene can be formulated as pharmaceutical compositions or combinations with a pharmaceutically acceptable carrier. The pharmaceutical compositions or combinations can be formulated to be administered parenterally, topically, orally, intrathecally or by local administration. The pharmaceutical compositions or combinations can be formulated in any way and can be administered in a variety of unit dosage forms depending upon the condition or disease and the degree of illness, the general medical condition of each patient, the resulting preferred method of administration and the like. In one embodiment, the pharmaceutical composition or combination can be administered intrathecally. For example, in embodiments, the pharmaceutical composition or combination can be administered in vivo via intrathecal injection.

[ 0042 ] Pharmaceutical compositions or combinations of this invention can be prepared according to any method known to the art for the manufacture of pharmaceuticals. Such drugs can contain sweetening agents, flavoring agents, coloring agents and preserving agents. A formulation can be admixtured with nontoxic pharmaceutically acceptable excipients which are suitable for manufacture. Formulations may comprise one or more diluents, emulsifiers, preservatives, buffers, excipients, etc. and may be provided in such forms as liquids, powders, emulsions, lyophilized powders, sprays, creams, lotions, controlled release formulations, tablets, pills, gels, on patches, in implants, etc.

[ 0043 ] In some embodiments, the methods described herein can include co-administration with one or more inhibitory nucleic acids targeting SOD1 or MMP9, a transgene encoding a nuclear PTEN interfering peptide, an ER stress inhibitor, a stress granule blocker or a miRNA biogenesis activator. [ 0044 ] In another aspect, the present disclosure provides a method of screening a drug protecting motor neuron subtpyes in ALS in vitro , comprising (i) providing a stem cell line harboring a transgene comprising a fALS gene and a reporter gene, (ii) culturing a drug with the cell line; (iii) directing differentiation of the cell to the motor neuron cells and expression; and (iv) determining the said drug is effective to protect motor neuron subtypes in ALS if a motor neuron subtype is generated from the stem cell line.

[ 0045 ] In some embodiments, the stem cell line is an embryonic stem cell line or iPSC cell line. In some embodiments, the stem cell line is human or mouse stem cell line.

[ 0046 ] In some embodiments, the fALS gene is C9orf72 mutation containing expanded GGGGCC repeats, FUS-R521C, FUS-P525R, FUS-R521H, FUS-Q210H, FUS-R514G, FUS- C1574G, SOD1-G93A, SOD1-A4V, SOD1-D90A, SOD1-D91A, SOD1-H46R, SOD1-A89V, SOD1-I113T, TDP43-A315T, TDP43-Q343R, TDP43-M337V, TDP43-N345K, TDP43-I383V or TDP43-M337V.

[ 0047 ] In some embodiments, the reporter is a double reporter. In some embodiments, the double reporter is Hb9::RFP and FoxPl::GFP.

[ 0048 ] In a further aspect, the present disclosure provides a method for diagnosis of motor neuron subtypes in ALS in a subject, comprising providing a biological sample and detecting the presence of one or more members of microRNA-l7~92 cluster or nuclear PTEN, wherein the presence of dysregulated microRNA-l7~92 members or nuclear PTEN is indicative of a diagnosis of motor neuron subtypes in ALS or a susceptibility to motor neuron subtypes in ALS in the subject.

[ 0049 ] Also provided herein are kits for use in diagnosing motor neuron subtypes in ALS or susceptibility to motor neuron subtypes in ALS in a subject, comprising one or more reagents for detecting the presence of one or more members of mi croRNA- 17-92 cluster or PTEN in a biological sample. In a particular embodiment, the kit comprises at least one contiguous nucleotide sequence that is substantially or complementary to one or more members of microRNA- 17-92 cluster or PTEN for detecting the presence of one or more members of microRNA- 17-92 cluster or PTEN. For example, the nucleic acids can comprise at least one sequence which is complementary (completely, partially) to microRNA- 17-92 or PTEN. In one embodiment, the one or more reagents in the kit are labeled, and thus, the kits can further comprise agents capable of detecting the label. The kit can further comprise instructions for detecting motor neuron subtypes in ALS using the components of the kit.

[ 0050 ] The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

EXAMPLES

[ 0051 ] MATERIALS AND METHODS

[ 0052 ] Mice

[ 0053 ] Mice carrying the mutant human SOD1G93A transgene (B6SJL-

Tg(SODl*G93A)lGur/J were purchased from Jackson Laboratory. The motor neuron-specific mir 17-92 -over expressing mice, OUg2c re/+ ; ROSA26-loxp-STOP-loxp- mir-17~92/f ( mir-17~92 MN-OE ), were generated previously (20), and were further crossed with SOD1G93A /+ ; ROSA26- loxp-STOP-loxp-mir-17~92f / f mice to obtain SODlG 93A ;Olig2cre /+ ; ROSA26-loxp-STOP-loxp-mir- 17~92/fforf /+ (ALS; mir-17~92 MN- OE ) rescue mice. For all experiments, ALS; mir-17~92 MN-OE mice were compared with their SOD1G93A ; Olig2cre /+ or SOD1G93A ; ROSA26-loxp-STOP-loxp- mir-17~92f /+ or f / fALS littermates. [ 0054 ] To generate mice with double transgenic (Tg) reporters, the spinal MN reporter Hb9::RFP construct was electroporated into BALB/c embryonic stem cells (ESCs), and injected into the host mouse. Several chimeric mice were bom and we further crossed them to C57BL/6J wild type mice to obtain the germ line-transmitted transgenic Hb9::RFP founder lines. The FoxPlr. GFP ES cell line that harbors a BAC with -195 kb of the 5' FoxPl sequence and GFP inserted at the initiating codon (a kind gift from Jeremy Dasen and Hynek Wichterle) (44) was used to derive the FoxPl ::GFP mouse strain in a C57BL/6 background. We then mated the FoxPlr. GFP line to the Hb9::PFP line to get mice with double Tg reporters (FoxP 1 : :GFP ; Hb9::RFP) in the C57BL/6 background. Double Tg FoxPlr. GFP; Hb9::RFP embryos were analyzed at E12.5 to validate the efficacy of the double reporters. All of the live animals were kept in an SPF animal facility, approved and overseen by IACUC Academia Sinica.

[ 0055 ] Tissue collection

[ 0056 ] Mice were sacrificed under anesthetic with 300 pL of 20 mg/mL Avertin (2,2,2- Tribromoethanol, Sigma) and subsequently perfused with PBS and 4% paraformaldehyde (PFA) in PBS. Whole spinal cords were isolated, refixed and sucrose cryoprotected. Spinal cords were embedded in FSC 22 frozen section media (Leica), and cut into 10 pm cryostat sections, which were then subjected to immunostaining or in situ hybridization by 3’ DIG-labeled LNA miRCURY probes (Exiqon). Detailed description of the procedures and analysis is provided in SI materials and methods.

[ 0057 ] Laser capture microdissection

[ 0058 ] Mice were sacrificed under anesthetic with Avertin and subsequently perfused by DEPC-PBS. The spinal cord was removed and dissected into several segments within 7 min, followed by embedding in FSC 22 frozen section media (Leica). The frozen blocks were cryosectioned at -20°C into 20 mih-thick slices and stored at -80°C until the laser capture procedure.

[ 0059 ] For the staining of MNs, each slice was washed by RNase-free 70% ethanol for 60 seconds, stained with 1% Azure B (MP Biomedicals) in washing solution for 180 seconds, followed by another 20 seconds-wash, and then air-dried completely. MNs were identified as large, darkly-stained cell bodies residing in the ventral lateral region of the L5 lumbar spinal cord, whereas dorsal non-MNs were identified as Azure B-stained cell bodies located in the dorsal spinal cord. Around 400-500 MNs or dorsal non-MNs were microdissected (PALM MicroBeam, ZEISS, 20X objective) from each mouse and collected into a single tube. MNs and dorsal non-MNs from 3-4 mice per strain were collected.

[ 0060 ] Spinal motor neuron counts

[ 0061 ] At each indicated stage, ChATon MNs in the L5 ventral horns with DAPI were counted on one side of a 10 pm sectioned spinal cord. MNs that did not show regular nuclear shapes were excluded. The histograms represent the average MN counts from at least 3 sections per mouse (n > 3 mice) of the same age and genotype.

[ 0062 ] AAV9 virus preparation and injection

[ 0063 ] The construction of AAV9-mir-l 7~92 plasmid was described in SI methods. AAV9- mir-17~92 and AAV9-GFP were packaged by the AAV Core Facility in Academia Sinica. For intrathecal injection, mice at P60 were anesthetized by isoflurane. The lumbar spinal cord was exposed through a ~l.5cm window surgically cut on the back of the mice. 20 pL of either AAV9-GFP or AAV9 -mir-17~92 (5xl0 9 vg/pL) was injected at a rate of 4 pL/min into the groove between the L6 and Sl segments using a 27G needle. A flick of the mouse’s tail indicated successful injection. Example 1 mirl 7-921 PTEN is expressed in adult spinal MNs and Requirement of mir-

17-92 to maintain MN survival

[ 0064 ] We discovered in a previous study (Tung, Y.T., Lu, Y.L., Peng, K.C., Yen, Y.P., Chang, M., Li, L, Jung, H., Thams, S., Huang, Y.P., Hung, J.H., et al. (2015). Mir-l7~92 Governs Motor Neuron Subtype Survival by Mediating Nuclear PTEN. Cell reports 11, 1305- 1318) that expression of the mir-17~92 miRNA cluster is enriched in developing LMC-MNs, and the cluster functions to promote the survival of LMC-MNs before they reach the innervating muscles from which they receive neurotrophic support. Mechanistically, mir-17~92 targets PTEN and its associated E3 ubiquitin ligase complex, i.e., Nedd4-2 and Ndfipl. As a consequence, PTEN is less mono-ubiquitinated and mostly remains in the cytoplasm to prevent LMC-MNs undergoing naturally-occurring apoptosis in the developing spinal cord (Figure 1 A).

[ 0065 ] Given that many spinal onset ALS patients start with limb weakness and that active denervation occurs more prominently in the limbs of ALS patients than in their thoracic paraspinal muscles, LMC-MNs appear to be more susceptible to undergoing degeneration compared to other MN types in ALS (Kanning, K.C., Kaplan, A., and Henderson, C.E. (2010). Motor neuron diversity in development and disease. Annual review of neuroscience 33, 409-440). This manifestation prompted us to examine if mir-17~92/nuclear PTEN (nPTEN) could account for the differential vulnerability of MN subtypes in ALS through its differential expression in adult MN subtypes (Figure 1A).

[ 0066 ] We firstly checked whether the expression of mir-17~92 remains enriched in adult spinal MNs by in situ hybridization (ISH) together with immunostaining in sectioned adult lumbar spinal cord at postnatal day 40 (P40), using miR-17 as the representative miRNA in the cluster and choline acetyltransferase (ChAT) as an MN marker (Figure 1B). Although miR-17 expression was detectable in neurons in the whole spinal cord, its expression was more prominent in the ventral lateral spinal cord (Figures 1B and S1A). Quantification verified that the expression level of miR-17 in spinal MNs was higher compared to Renshaw cells (RCs) locating at the ventral spinal cord.

[ 0067 ] To corroborate the enrichment of each member of the mir-17~92 cluster in MNs, we laser microdissected the cell bodies of ventral lateral MNs and non-MNs from dorsal spinal cords (Figures 1C and 1D) and performed quantitative RT-PCR (qPCR). ChAT was used to ensure that we captured bona fide MNs (Figure 1E). Compared to non-MNs from dorsal spinal cord, the expression of mir-17~92 was more abundant in MNs of adult mouse spinal cord (Figure 1F), supporting that mir-17~92 expression is enriched and sustained from developing embryos to adult spinal MNs.

[ 0068 ] Next, we examined the expression of the target of mir-17~92 (i.e., PTEN) in adult spinal cord. Interestingly, PTEN was abundantly expressed in ventral -lateral ChAT on MNs and was predominantly localized in the cytosol, whereas expression of PTEN in dorsal cells and Calbindin 011 RCs was nearly undetectable. These results indicate that in the adult spinal cord, PTEN is expressed preferentially in the spinal MNs and exhibits a discriminatory cytosolic localization, possibly a reflection of the enrichment of mir-17~92 expression.

[ 0069 ] Our previous study indicated that mir-17~92 deletion in MNs (Olig2 Cre/+; mir- 17-92 fl oxed = m ir-17 92 mnd ) led to specific loss of LMC-MNs at embryonic stage E l 2.5—13.5 (Tung, Y.T., Lu, Y.L., Peng, K.C., Yen, Y.P., Chang, M., Li, T, Jung, H., Thams, S., Huang, Y.P.,

Hung, J.H., et al. (2015). Mir-l7~92 Governs Motor Neuron Subtype Survival by Mediating Nuclear PTEN. Cell reports 77, 1305-1318). Consequently, ~ 70% of the mir-17 92 MND embryos died as neonates, possibly due to the severe movement defects (N = 69, Table Sl). The remainder of the surviving mir-17 ~92 MND mice allowed us to assess the MN phenotype at later stages after birth. Among these mutants (N =17), -85% exhibited smaller size and reduced body weight. Nearly all mir-17 ~92 MND mice displayed some hallmarks of “ALS” pathologies, including that 1) ChAT on MN numbers were significantly reduced in the spinal cords of the mir- ig~92 MND mice at P30 (Figure 1G and quantification in II). 2). Remarkably, no denervation was detected at P30 in the gastrocnemius (GA) muscle of the control mice, whereas the mir- mice manifested drastic denervation of motor endplates (Figure 1H and quantification in 1J). 3). The compound muscle action potential (CMAP) in mir-17 ~92 MND mice was reduced by - 35% relative to control values (Figure 1K). Taken all together, our analyses indicate that mir-17-92/PTEN is highly expressed in adult spinal MNs and the down-regulation of mir-17~92 in MNs is sufficient to cause MN loss.

Example 2 Early presymptomatic dysregulation of mir-17~92/nPTEN axis in MNs of S0D1 G93A mice

[ 0070 ] To test the hypothesis that dysregulation of mir-17~92/nPTEN pathway is involved in MN degeneration of ALS, we used S0D1 G93A mouse model as a paradigm. Throughout adulthood, numbers of ChAT on MNs remained unaffected in S0D1 G93A lumbar spinal cords before P80 and were slightly reduced at P100 (Figure 2A, quantification in 2B). Even with comparable MN numbers between Ctrl and S0D1 G93A mice at P80, we already observed a significant down-regulation of miR-17 expression in S0D1 G93A MNs (Figure 2C, quantification in 2D). The down-regulation trend appeared more prominently in MNs as miR- 17 expression remained low and comparable in both dorsal Tuj l on interneurons (dlNs, resistant to ALS) and calbindin 011 RCs (intact in presymptomatic ALS stages) (Wootz, FL, Fitzsimons- Kantamneni, E., Larhammar, M., Rotterman, T.M., Enjin, A., Patra, K., Andre, E., Van Zundert, B., Kullander, K., and Alvarez, F.J. (2013). Alterations in the motor neuron-renshaw cell circuit in the Sodl(G93A) mouse model. J Comp Neurol 527, 1449-1469) throughout the indicated ages between both Ctrl and S0D1 G93A mice.

[ 0071 ] To precisely quantify the expression of miR-17 and other members of mir-17~92 cluster in intact S0D1 G93A lateral MNs before degeneration, we used laser microdissection to collect MNs with healthy morphology in the lateral ventral spinal cord based on their size and shape (see STAR Methods for details), from both Ctrl and S0D1 G93A mice at P100 (Figure 2E) and compared mRNA/miRNA expression by qPCR. While the expression of ChAT was unchanged between the microdissected control and S0D1 G93A MNs (Figure 2F), several members of mir-17~92 had already manifested significantly declined expression in S0D1 G93A MNs (Figure 2G). These data reflected that mir-17~92 expression is down-regulated prior to onset of MN death in S0D1 G93A MNs.

[ 0072 ] Moreover, along with the down-regulation of mir- 17-92 in the same sets of microdissected MNs, we observed a concomitant increase in the expression of its targets PTEN , Nedd4-2 and Ndfipl in captured S0D1 G93A MNs at P100 when compared to controls (Figures 2H). As Nedd4-2/Ndfipl promote PTEN mono-ubiquitination to facilitate its nuclear import (Tung et al., 2015), we next analyzed subcellular PTEN localization in MNs from control and S0D1 G93A mice at P40, P80 and P100. In S0D1 G93A MNs at P100, the intensity of PTEN increased significantly in the whole cell (PTEN total ), as well as in the nucleus (PTEN nucleus ) (Figure 21). Additionally, we also observed a dramatically elevated ratio of PTEN nucleus/cytoso1 in S0D1 G93A MNs (Figure 2J), indicating that the elevated PTEN tended to accumulate in the MN nuclei. The delay until significant nuclear PTEN accumulation (P100) upon mir- 17-92 reduction (P80) might reflect the time required for coupling of PTEN post-transcriptional regulation and post-translational modification. In contrast to ChAT on ventral lateral MNs, PTEN expression in control, S0D1 G93A Calbindin 011 RCs and Tuj l on dINs remained nearly undetectable at all ages (data not shown). Collectively, the data demonstrates that disruption of the mir-17~92/nPTEN pathway is a signature event in spinal MNs before the onset of MN loss in the S0D1 G93A spinal cord.

Example 3 Selective dysregulation of mir-17~92 in S0D1 G93A LMC-MNs

[ 0073 ] To further examine if mir- 17-92 dysregulation contributes to the vulnerability of limb-innervating MNs to ALS, in this scenario, we established a novel double transgenic mouse strain in which limb-innervating LMC-MNs are labeled with GFP ( Foxpl ::GFP ) and generic motor neurons are labeled with RFP ( Hb9::RFP ). To verify the fidelity of the reporter, we examined the expression pattern of the double fluorescence reporters along the rostrocaudal axis of the spinal cord. As expected, Foxpl ::GFP signal was detected in LMC-MNs, whereas Hb9::RFP signal was detected in both LMC-MNs and non-LMC-MNs. Accordingly, LMC-MNs and non-LMC-MNs could be distinguished by a combination of the transgenic reporters in vivo , with GFP on /RFP on representing LMC-MNs and GFP off /RFP on representing non-LMC-MNs. However, Foxpl ::GFP became less specifically expressed in LMN-MNs and was expressed in some spinal INs. Moreover, Hb9::RFP was also restricted to subsets of adult MNs (data not shown). Consequently, we adopted an alternative embryonic stem cell (ESC) differentiation approach by taking advantages of the fact that: 1) high-fidelity transgenic reporters are available to delineate MN subtypes; 2) conditions for deriving MN subtypes with authentic rostrocaudal spinal identities are well established (Davis-Dusenbery, B.N., Williams, L.A., Klim, J.R., and Eggan, K. (2014). How to make spinal motor neurons. Development 14J 491-501 Davis- Dusenbery et ah, 2014); 3) postmitotic MN subtypes can be FACS-collected with a dual reporter system; and 4) conditions for maturing and accelerating MN degeneration in vitro are well defined (Thams, S., Lowry, E.R., Larraufie, M.H., Spiller, K.J., Li, H., Williams, D.J., Hoang, P., Jiang, E., Williams, L.A., Sandoe, J., et al. (2018). A Stem Cell-Based Screening Platform Identifies Compounds that Desensitize Motor Neurons to Endoplasmic Reticulum Stress. Molecular therapy : the journal of the American Society of Gene Therapy).

[ 0074 ] In this scenario, we further derived Foxpl ::GFP; Hb9::RFP embryonic stem cell (ESC) line and then differentiated them into spinal MNs using a defined protocol that generates cervical/brachial Hoxa5 0n MNs (composed of -15% LMC-MNs and -85% non-LMC-MNs). Immunostaining of ESC-derived MNs revealed that Hb9::RFP cells are co-expressed with the generic MN markers Isl 1/2 and Hb9, whereas Foxpl ::GFP cells were manifested with the LMC- MN identity as Foxpl 0n /Lhx3 0ff . This outcome was further validated by qPCR analysis from FACS-purified GFP on /RFP on cells, which exhibited high Raldh2 and Foxpl expression, but low expression of Lhx3. Above all, our double reporter system provides an ideal platform to study LMC-MN versus non-LMC-MN phenotypes in a well-defined differentiation system.

[ 0075 ] Next, we crossed the Foxpl ::GFP; Hb9::RFP strain to Ctrl and S0D1 G93A mice to get the Ctrl and S0D1 G93A double reporter ESC lines, then harnessed them into MN subtypes (Figure 3A). In the absence of stress conditions upon differentiation, both Ctrl and S0D1 G93A lines could be differentiated into comparable LMC-MNs and non-LMC-MNs with no preferential cell loss. The expression levels of mir-17~92 members were also similar between Ctrl and S0D1 G93A lines in the basal condition. To accelerate ALS disease progression in vitro , we applied cyclopiazonic acid (CPA) to Ctrl and S0D1 G93A ESC-derived MNs. In a recent study that applied the same ESC differentiation approach used here (Thams, S., Lowry, E.R., Larraufie, M.H., Spiller, K.J., Li, H., Williams, D.J., Hoang, P., Jiang, E., Williams, L.A., Sandoe, J., et al. (2018). A Stem Cell-Based Screening Platform Identifies Compounds that Desensitize Motor Neurons to Endoplasmic Reticulum Stress. Molecular therapy : the journal of the American Society of Gene Therapy), CPA was demonstrated to be an endoplasmic reticulum stressor that exhibited preferential toxicity to MNs and only mild effects on INs. Upon CPA stress, we found that non-LMC-MN populations were more resistant to the ER stressor in both Ctrl and S0D1 G93A MNs. In contrast, S0D1 G93A LMC-MNs exhibited drastic cell loss after 24-48 hours of CPA treatment compared to Ctrl LMC-MNs (Figures 3B and 3C). Notably, no differential degeneration was displayed between Ctrl and S0D1 G93A ESC-derived MNs when treated with vehicle (DMSO) alone (Figures 3B and 3C). Thus, using our newly established ALS double reporter system, we could capture the differential degrees of degeneration among MN subtypes under CPA treatment.

[ 0076 ] To elucidate the differential sensitivity to CPA stress in MN subtypes, we investigated the expression of mir-17~92 and its targets (. PTEN , Ndfipl and Nedd4-2) in our ESC-derived MN system at 24 hours post-CPA treatment (i.e. at a time-point prior to LMC-MN loss). In S0D1 G93A LMC-MNs, we observed a trend of down-regulation of mir-17~92 members, whereas there was no significant difference being observed between Ctrl and S0D1 G93A non- LMC-MNs (Figures 3D and 3E). Concomitantly, expressions of the mir-17~92 targets (. PTEN , Ndfipl and Nedd4-2) were also specifically up-regulated in S0D1 G93A LMC-MNs (Figures 3F and 3G). Above all, S0D1 G93A LMC-MNs are more prone to undergoing degeneration upon stress, with a selective manifestation of mir-17~92 downregulation.

Example 4 SOD1G93A LMC-MNS are more prone to mir-17~92/nPTEN dysregulation

[ 0077 ] Next, we investigated changes in the subcellular localization of PTEN in LMC-MNs and non-LMC-MNs derived from both Ctrl and S0D1 G93A mice at 24 hours post-CPA treatment. PTEN was mainly distributed in the cytosol in the Ctrl sets of both non-LMC-MNs and LMC- MNs (Figure 4A), similar to our observations of adult spinal MNs (Figure 21). In accordance with our model, we observed a significant increase in the PTEN nucleus/cytoso1 ratio in S0D1 G93A LMC-MNs upon stressor treatment compared to that of Ctrl LMC-MNs, whereas there was no difference in the PTEN nucleus/cytoso1 ratio of Ctrl and S0D1 G93A non-LMC-MNs (Figure 4A; quantification in Figure 4B).

[ 0078 ] To further scrutinize if MN subtypes respond differentially to the cytotoxicity of nuclear PTEN, we adopted in ovo system by electroporation of PTEN fused with either nuclear localization or nuclear export signals (NLS and NES) into developing chicken embryos (Figure 4C). Compared to marginal effects of PTEN-WT and PTEN-NES, ectopic overexpression of PTEN-NLS instead led to a significant loss of Foxpl 0n LMC-MNs. Interestingly, Lhx3 on non LMC-MNs are more resistant to PTEN expression. This result indicates that nPTEN preferentially causes LMC-MN death (Figure 4D, quantification in Figure 4E).

[ 0079 ] Taken all together, these findings suggest that selective mir-17~92/nPTEN dysregulation occurs early in S0D1 G93A LMC-MNs and confers the MN subtype vulnerability of ALS.

Example 5 Recapitulation of hsa-mir-l 7~92 /nPTEN dysregulation in human ALS S0D1 +/L144F iPSC-derived MNs

[ 0080 ] To further explore whether the role of mir-17~92 is relevant in a human MN context, we differentiated a human ESC line harboring the MN reporter Hb9::GFP (HuES3 HB9::GFP ) into MNs under defined conditions (Maury, Y., Come, T, Piskorowski, R.A., Salah-Mohellibi, N., Chevaleyre, V., Peschanski, M., Martinat, C., and Nedelec, S. (2014). Combinatorial analysis of developmental cues efficiently converts human pluripotent stem cells into multiple neuronal subtypes. Nature biotechnology) that recapitulate the developmental temporal order from OLIG2 on progenitor MNs (day 9) into HB9 on /ISLl on nascent MNs at day 14. Hb9::GFP on MNs were FACS-collected at day 16 and the enrichment of the LMC-MN markers were authenticated by FOXP1 and RALDH2 expressions. Similar to our finding in mouse MNs (Figure 1F), hsa-mir- 17-92 was expressed more abundantly in FACS-purified HB9::GFP on human MNs.

[ 0081 ] Next, we acquired human induced pluripotent stem cells (iPSCs) from ALS patients carrying the S0D1 +/L144F mutation (Boulting, G.L., Kiskinis, E., Croft, G.F., Amoroso, M.W., Oakley, D.H., Wainger, B.J., Williams, D.J., Kahler, D.J., Yamaki, M., Davidow, L., et al. (2011). A functionally characterized test set of human induced pluripotent stem cells. Nature biotechnology 29, 279-286), and further generated an isogenic control SOI) I line mediated by CRISPR-Cas9 system to correct the mutated DNA sequence (Figures 5 A and 5B). Both of these iPSC lines exhibited indistinguishable pluripotent hallmarks with normal karyotypes under our culture conditions, and SOD1 expression levels were comparable between the two lines. Both iPSC lines could be successfully differentiated into SMI32 on ISLl on MNs with -80% efficiencies following the developmental temporal order. Dissociated MNs were then cultured in neurotrophic factor (NF)-supplemented medium for another two weeks until manifesting a more matured marker CHAT. The majority (-75%) of ISLl on MNs acquired FOXPl on LMC-MN identity at day 16 and thereafter, providing an ideal system to study human LMC-MNs in ALS content.

[ 0082 ] To accelerate ALS progression, we withdrew NFs in the media after MN maturation (from day 32-60) (Shi, Y., Lin, S., Staats, K.A., Li, Y., Chang, W.H., Hung, S.T., Hendricks, E., Linares, G.R., Wang, Y., Son, E.Y., et al. (2018). Haploinsufficiency leads to neurodegeneration in C90RF72 ALS/FTD human induced motor neurons. Nature medicine 24 , 313-325), resulting in a more prominent reduction of FOXPl on ISLl on LMC-MNs derived from ALS-S0D1 +/L144F iPSCs (Figures 5C-5E). Next, we checked hsa-mir- 17~92/nPTEN levels before and after 14 days of NF withdrawal, a stage before a severe loss of ALS-MNs, reminiscent to the onset of MN reduction in vivo. Indistinguishable expression of hsa-mir- 17-92, PTEN and NDFIP1 protein levels, as well as nuclear-to-cytosolic ratio of PTEN in Ctrl -SOD1 +/+ and ALS- S0D1 +/L144F MN cultures were observed before NF withdrawal. However, upon 14 days after NF withdrawal, there was a drastic reduction of the expression of hsa-mir-17~92 (Figure 5F), concomitant with increased protein levels of PTEN, NEDD4-2 and NDFIP1, as well as enhancement of PTEN nucleus/cytoso1 ratio in ALS-S0D1 +/L144F MN culture (Figures 5G and 5H). Notably, we did not observe significant difference in cell loss between the Ctrl -SODl +/+ and ALS-S0D1 +/L144F lines at this stage (Figure 5E). This finding corroborated the observations in mouse S0D1 G93A model: the down-regulation of hsa-mir-17~92 with nuclear PTEN accumulation is exhibited prior to LMC-MN loss in SOD1-ALS type.

Example 6 Targeting hsa-mir-l 7~92 for therapeutic application in human S0D1 +/L144F iPSC-derived MNs

[ 0083 ] To exploit if mir-17~92 could serve as a target to spare MN degeneration in ALS, we then tested if manipulation of mir-17~92 expression could rescue MN degeneration in human ALS-iPSC derived MNs. We utilized lentivirus (LV) to deliver hsa-mir-17~92 or YFP as a control into iPSC~MNs before NF withdrawal (Figure 6A). The persistent overexpression of YFP and hsa-mir-17~92 in ALS-SODI / / AINs throughout the survival assay were confirmed by immunostaining and qPCR, respectively (Figures 6B). As expected, ectopic hsa-mir-17~92 expression led to the repression of PTEN, NEDD4-2 and NDFIP1 in ALS-SODI l441' MNs (Figure 6C), with notable rescued effects on of ALS-SODI / / LMC-MN populations (Figure 6D and quantification in 6E). Above all, these data supported that overexpression of hsa-mir- 17-92 in MN context can relieve MN degeneration in ALS-S0D1 +/L144F , thereby providing proof-of-principle evidence that hsa-mir-17~92 could be an ideal therapeutic target for ALS treatment.

Example 7 Amelioration of ALS symptoms in S0D1 A mice by overexpressing mir- 17-92 in MNs

[ 0084 ] To exploit if mir-17~92 could rescue MN degeneration in ALS in vivo , we then tested if manipulation of mir-17~92 expression could ameliorate ALS symptoms in S0D1 G93A mice. We have previously generated conditional mir-17~92 MN E ( Olig2 Cre/+ ; ROSA-26-LSL-mir- 17-92 N) mice, and these mice were healthy and exhibited a normal lifespan (Tung, Y.T., Lu, Y.L., Peng, K.C., Yen, Y.P., Chang, M., Li, L, Jung, H., Thams, S., Huang, Y.P., Hung, J.H., et al. (2015). Mir-l7~92 Governs Motor Neuron Subtype Survival by Mediating Nuclear PTEN. Cell reports 77, 1305-1318). Here, we further crossed mir-17~92 MN E mice to the S0D1 G93A strain in a pure C57BL/6 background (Figure 7A). Restoration of mir-17~9 expression in ChAT on MNs was validated by in situ hybridization together with immunostaining of lumbar spinal cord sections of [S0D1 G93A ; mir-17~92 ' /v / ] mice. Compared to the barely detectable level of miR-17 expression in S0D1 G93A siblings at P100, [S0D1 G93A mir- 17-92 ' /v_ / ] mice retained high levels of miR-17 expression. As a consequence, the reduced MN numbers in S0D1 G93A mice was significantly spared in [S0D1 G93A mir-17~92 ' /v_ / ] mice at P 100 (Figure 7B, quantification in 7C). At a molecular level, the elevated PTEN nucleus/cytoso1 ratio in S0D1 G93A MNs was reduced upon mir-17~92 overexpression (Figure 7D, quantification in 7E).

[ 0085 ] To further examine whether mir-17~92 overexpression improves ALS symptoms and motor function, we performed a series of physiological assays (Kanning, K.C., Kaplan, A., and Henderson, C.E. (2010). Motor neuron diversity in development and disease. Annual review of neuroscience 33, 409-440). [ S0D1 G93A ; mir-17~92 ' /v_ / ] mice gained body weight and exhibited alleviated hindlimb clasping symptoms at P140. The kyphosis phenotype caused by muscle weakness in S0D1 G93A mice was also ameliorated by mir-17~92 overexpression (examined by roentgenogram) (Figure 7F). Behaviorally, [S0D1 G93A ; mir-17~92 ' /v_ / ] mice performed better in a rotarod test (Figure 7G). Strikingly, median survival of S0D1 G93A mice in our C57BL/6 colony was -160 days, but this increased to -182 days on overexpression of mir-17~92 , representing a -14% increase in lifespan (Figure 7H).

Example 8 Adult administration of AAV9 -mirl 7~92 prolongs S0D1 G93A mice survival

[ 0086 ] The promising outcome of overexpressing mir-17~92 in S0D1 G93A mice prompted us to test the potential of mir-17~92 as a therapeutic measure in adult mice. We utilized self- complementary adeno-associated vector serotype 9 (scAAV9) to overexpress mir-17~92 in the whole spinal cord by intrathecal injection at P60 (Foust, K.D., Salazar, D.L., Likhite, S., Ferraiuolo, L., Ditsworth, D., Ilieva, FL, Meyer, K., Schmelzer, L., Braun, L., Cleveland, D.W., et al. (2013). Therapeutic AAV9-mediated suppression of mutant SOD1 slows disease progression and extends survival in models of inherited ALS. Molecular therapy : the journal of the American Society of Gene Therapy 27, 2148-2159), which bypassed the potential function of mir-17~92 to promote ectopic proliferation of embryonic MN progenitors (Figures 8A). To test the efficiency of virus infection, we first injected scAAV9-GFP to Ctrl mice and observed strong and sustained GFP expression in spinal MNs as well as in some dorsal cells at 40 days post- injection. Then, scAAV9-mir-17~92 was injected in parallel to scAAV9-GFP into Ctrl and S0D1 G93A mice. We verified robust induction of exogenous mir-17~92 in the spinal cord by in situ hybridization of miR-17, as well as qPCR quantification of mir-17~92 members in the lumbar spinal cord (Figures 8B and 8C). [ 0087 ] To evaluate the mice in a more clinically relevant setting, we assayed MN and gastrocnemius (GA) muscle connectivity by measuring CMAP. We performed CMAP at several time points: from P60 (right before the AAV treatment) to P140. S0D1 G93A mice had just begun to manifest MN denervation at P60 and exhibited a slightly compromised CMAP amplitude. At P120 and P140, the CMAP response had significantly ameliorated upon ANV9-mir-17~92 treatment, but had not yet improved to the level of Ctrl mice (Figure 8D). Unlike how SOD1 antisense oligonucleotide (ASO) treatment promotes MN reinnervation (McCampbell, A., Cole, T., Wegener, A.J., Tomassy, G.S., Setnicka, A., Farley, B.J., Schoch, K.M., Hoye, M.L., Shabsovich, M., Sun, L., et al. (2018). Antisense oligonucleotides extend survival and reverse decrement in muscle response in ALS models. J Clin Invest 725, 3558-3567), our data suggests that overexpression of mir-17~92 enhances MN survival significantly rather than promotes MN reinnervation. Finally, and most importantly, adult delivery of mir-17~92 also prolonged the lifespan of S0D1 G93A mice from a median of 161 days to 184 days (Figure 8E).

[ 0088 ] Taken all together, our results support that mir-17~92 is a sine qua non intrinsic regulator to sustain MN survival via regulating PTEN subcellular translocation in adult MNs. In the ALS content, overexpression of mir-17~92 in MNs by either a genetic approach or scAAV9 delivery in adults delays the onset of MN degeneration, enhances motor function, and increases the lifespan of our ALS mouse model. These findings envisage mir-17~92 to be a prognosis marker for MN degeneration and a promising candidate therapeutic target for ALS patients.